Nicotinamide nucleotides play a major role in biological oxidations; reductive synthesis of carbohydrates, lipids and amino acids; the various monooxygenase reactions involved in detoxification and steroid hydroxylation reactions; energy production; and metabolic regulation. Nicotinamide nucleotide transhydrogenases are potential control devices for maintaining the cellular balance of NAD(H) and NADP(H), and thereby contributing to the regulation of various metabolic reactions linked to these nucleotides. The mitochondrial transhydrogenase system is of particular interest, because the equilibrium of the mitochondrial nicotinamide nucleotide pool can be shifted in favor of NADPH production in an endergonic reaction, which utilizes stoichiometric amounts of ATP. The objectives of the program are (1) to study the molecular mechanisms of nonenergy-linked (NADPH yields NAD) and energy-linked (NAD(P)H yields NADP) transhydrogenation; (2) to develop improved procedures for purification of highly active enzyme in good yield; (3) to investigate the mechanisms of energy transfer to the enzyme, and utilization by the transhydrogenase reaction; and (4) as a long-term goal to relate these findings to studies on (a) the general mechanism of energy conservation, transduction and transfer by mitochondria, and (b) the physiological role of the mitochondrial transhydrogenase reaction. Our studies made possible by this grant have provided considerable clarification regarding energy-linked and nonenergy-linked transhydrogenation, and have allowed us to formulate a general mechanism for these reactions.